3-methyladenine antagonizes the anti-hepatic fibrosis effect of betulinic acid by inhibiting the autophagy pathway
3-甲基腺嘌呤通过抑制自噬通路拮抗白桦脂酸抗肝纤维化效应的机制研究

摘要

目的 探讨3-甲基腺嘌呤（3-Methyladenine, 3-MA）是否通过抑制自噬加重CCl₄诱导的小鼠肝纤维化。方法 将50只SPF级雄性C57BL/6小鼠随机分为对照组、模型组、BA（Betulinic acid，白桦脂酸）组、3-MA处理组、3-MA+BA组，每组10只。除对照组小鼠给予1.5ml/kg•d纯橄榄油腹腔注射外，余下各组小鼠均按四氯化碳橄榄油溶液（25%，CCl4∶橄榄油=1∶3），2ml/kg•d腹腔注射，构建肝纤维化模型，造模第1周起同步给予干预药物，3-MA处理组及3-MA+BA组在腹腔注射CCl₄橄榄油溶液前2小时，每日1次腹腔注射3-MA（10mg/kg）；BA组及3-MA+BA组在CCl4橄榄油溶液腹腔注射半小时后给予白桦脂酸30mg/kg的悬浊液灌胃，持续干预6周。HE染色评估肝组织病理损伤，天狼星红苦味酸染色检测胶原纤维沉积；Western blot检测肝星状细胞活化标志物α-SMA的表达水平。结果 与对照组相比，3-MA组小鼠血清ALT、AST水平显著升高（P<0.05），3-MA组小鼠的肝组织较模型组的肝细胞炎症反应明显，肝脏红色胶原纤维沉积较模型组增多，α-SMA蛋白表达上调；3-MA+BA组血清ALT、AST水平较3-MA组显著降低（t=12.62，P<0.05；t=12.42，P<0.05），仅有少量的炎症细胞在汇管区聚集，细胞水肿及空泡变性较3-MA组减轻，胶原纤维沉积减少α-SMA蛋白表达介于3-MA组和BA组之间；BA组较3-MA+BA组血清中ALT、AST水平明显降低，病理改变明显减轻，细胞结构较完整，无明显细胞水肿及空泡变性，仅汇管区有微量胶原纤维，α-SMA蛋白表达较模型组明显减少。结论 3-MA通过抑制自噬显著加重CCl₄诱导的小鼠肝纤维化，拮抗白桦脂酸抗肝纤维化的作用。

Abstract

Objective To investigate whether 3-methyladenine (3-MA) exacerbates CCl₄-induced liver fibrosis in mice by inhibiting autophagy, thereby antagonizing the anti-fibrotic effect of betulinic acid (BA).
Methods Fifty SPF-grade male C57BL/6 mice were randomly divided into a control group, a model group, a betulinic acid group, a 3-MA treatment group, and a 3-MA+BA group, with 10 mice in each group. Except for the control group, which received intraperitoneal injections of 1.5 ml/kg•d of pure olive oil, all other groups underwent intraperitoneal injections of a 25% carbon tetrachloride (CCl₄) and olive oil solution (CCl₄: olive oil = 1:3) at 2 ml/kg•d to establish a liver fibrosis model. Interventional drugs were administered simultaneously from the first week of model establishment. The 3-MA treatment group and the 3-MA+BA group received intraperitoneal injections of 3-MA (10 mg/kg) once daily 2 hours before the intraperitoneal injection of the CCl₄ and olive oil solution. The BA group and the 3-MA+BA group received gavage with a 30 mg/kg suspension of betulinic acid half an hour after the intraperitoneal injection of the CCl₄ and olive oil solution, with continuous intervention for 6 weeks. Liver histopathological damage was assessed using HE staining, collagen fiber deposition was detected using Sirius red-picric acid staining, and the expression level of α-smooth muscle actin (α-SMA), a marker of hepatic stellate cell activation, was detected by Western blot.
Results Compared with the control group, the serum levels of ALT and AST in the 3-MA group were significantly increased (P<0.05). The liver tissue of the 3-MA group exhibited more pronounced inflammatory responses in hepatocytes and increased deposition of red collagen fibers in the liver compared with the model group, with upregulation of α-SMA protein expression; The levels of serum ALT and AST in the 3-MA+BA group were significantly lower than those in the 3-MA group (t=12.62, P<0.05; t=12.42, P<0.05). Only a small number of inflammatory cells accumulated in the portal area, and cell edema and vacuolar degeneration were less severe than in the 3-MA group. Collagen deposition was reduced, and α-SMA protein expression was between that of the 3-MA group and the BA group. The levels of serum ALT and AST in the BA group were significantly lower than those in the 3-MA+BA group, and the pathological changes were significantly alleviated. The cell structure was relatively intact, with no obvious cell edema or vacuolar degeneration. Only a small amount of collagen fibers were present in the portal area, and α-SMA protein expression was significantly reduced compared to the model group.
Conclusion   3-MA significantly exacerbates CCl₄-induced liver fibrosis in mice by inhibiting autophagy, antagonizing the anti-fibrotic effect of betulinic acid.